Porous adsorptive or chromatographic media

ABSTRACT

A porous substrate capable of adsorptive filtration of a fluid having a porous self-supporting substrate and one or more porous, adsorptive polymeric coatings comprising from about 1 to about 80% of the void volume of the pores of the substrate. The resultant substrate has good convective and diffusive flow and capacity. The substrate may be cross-linked, have one or more capture chemistries attached to it and is useful as a chromatography media for the selective filtration of desired species including biomolecules such as proteins and DNA fragments.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/548,462, filed on Feb. 27, 2004, and of U.S. Provisional ApplicationNo.: 60/542,025, filed on Feb. 5, 2004.

BACKGROUND OF THE INVENTION

Chromatography is a general separation technique that uses thedistribution of the molecules of interest between a stationary phase anda mobile phase for molecular separation. The stationary phase refers toa porous media and imbibed immobile solvent. Columns with associated endcaps, fittings and tubing are the most common configuration, with themedia packed into the tube or column. The mobile phase is pumped throughthe column. The sample is introduced at one end of the column, and thevarious components interact with the stationary phase and are adsorbedto or in the media or traverse the column at different velocities. Theseparated components are collected or detected at the other end of thecolumn. Adsorbed components are released in a separate step by pumpingan eluant solvent through the column. Chromatographic methods includedamong other methods, gel chromatography, ion exchange chromatography,hydrophobic interaction chromatography, reverse phase chromatography,affinity chromatography, immunoadsorption chromatography, lectinaffinity chromatography, ion affinity chromatography and other suchwell-known chromatographic methods. Current “state of the art”chromatographic or adsorptive separations use bead-based, monolith ormembrane media to accomplish the desired separation. These threetechnologies (beads, monoliths and membranes) accomplish separations viadiffering physical forms and therefore operate in phenomenologicallydifferent ways. A major difference between these three media is therelationship between the adsorbing surface (where adsorption of anentity to a ligand or ligands occurs) and the convective fluid flow.

Bead based media have convective flow occurring at the bead surfacewhile most of the adsorbing surface is internal to the bead and can onlybe reached via diffusion. The convective fluid flow properties aredetermined by the bead size. Smaller beads require higher pressure toattain equivalent flow in a column. However, the equilibrium adsorbingcapacity is not determined by the bead size. Therefore, the staticcapacity and the flow properties of the materials are not necessarilycoupled or interdependent. However, because most of the capacity isaccessed through diffusion, the dynamic binding capacity (capacity in aflow-through mode at a given flow rate) is coupled to the bead size andtherefore to the convective flow properties of the adsorbent.

Typically in the area of chromatographic separations, polysaccharidepolymers, such as agarose, are used to make gel media by thermally phaseseparating the polymer from an aqueous solution. This can be donebecause these polymers have a melting point and a gel point. To processagarose for example, the polymer must be heated above its meltingtemperature, which is about 92° C., and dissolved in the presence ofwater. At or above that temperature, the polymer melts and the moltenpolymer is then solvated by water to form a solution. The polymerremains soluble in water as long as the temperature is above thepolymer's gel point, which is about 43° C. At and below the gel point,the polymer phase separates and becomes a hydrogel that takes onwhatever shape the solution was just before gelling. Additionally, asthe agarose approaches its gel point, the viscosity of the solutionbecomes higher and higher as the hydrogel begins to form.

Traditionally, for polysaccharide beads, such as are used inchromatography media, the heated solution is kept above its gel pointand it is stirred into an immiscible, heated fluid, such as mineral orvegetable oil, to form beads. The two-phased material (beads of agarosein the immiscible fluid) is then cooled and the beads are recovered. Thebeads themselves are diffusionally porous and can then be used as madefor size exclusion chromatography. Preferably, they are furtherprocessed by crosslinking, the addition of various capture chemistriessuch as affinity chemistries or ligands, positive or negative charge,hydrophobicity or the like or combinations of crosslinking andchemistries to enhance their capture capabilities.

The beads are then loaded into a chromatography column forming a bed ofmedia through which a fluid containing the material to be captured ispassed. The beads are then washed to remove unbound contaminants andthen the captured material is eluted from the beads and collected.

Several problems exist with this type of media. The packing of the beadsinto a column is a difficult and laborious task. One needs to be surethat the column is properly packed so as to avoid channeling, bypass andblockages within the column. Packing of columns is often considered asmuch an art as it is a science.

The use of beads limits the depth of the media in process applicationsbecause of the pressure that must be overcome. Excess pressure maycompress the beads or require expensive pressure retaining capacity forthe column. Softer beads tend to compress more than rigid beads.Compression is indicated by a steep increase in pressure drop across thebed at sufficiently high flow rates. High pressure drop is due tocompression of the beads and subsequent reduction of void volume in asmall zone near the column outlet. The cumulative drag force of theflowing liquid through the bed causes compression. Drag force increaseswith higher flow rates, resulting in higher flow resistance and with bedheight. One often needs to run a soft gel bead system at a slower ratein order to ensure that the pressure drop is within acceptable bounds.

As the beads are porous and the selected molecule to be captured mustdiffuse into the pores of the media to be captured, the speed andcapacity of the system are diffusionally limited. There are twodiffusional limitations, one surrounding the bead where a film ofmaterial may form and inhibit movement of the selected molecule to thesurface of the bead and a second internal diffusional resistance whichis determined by the size, number and length of the pores formed in thebead surface. Additionally, the permeability of the media is related tobead size (which can vary widely) as well as the media stability. Largerbeads and beads with larger pores tend to have higher permeability.Beads that are not subject to or less subject to compression (by theweight of the beads above them coupled with the pressure under which thefluid flows through the bed) also tend to have greater permeability.However, at high flow rates, permeability does decrease and dynamiccapacity also decreases.

An alternative has been to use membrane or monolithic adsorbers. Formembrane and monolithic media, the convective flow is directly incontact with absorbing surface. Absorbing entities do not have to relyon diffusion to reach the absorbing surface. Because the convective flowis in direct contact with the absorbing surface in monolithic andmembrane media, the fluid flow and absorbing capacity are coupled. Forexample, the surface area of a membrane decreases with increasingaverage pore size. Because the binding capacity is only a surfacephenomenon in this design, as the pore size increases the bindingcapacity decreases. However, one advantage of this surface dominatedbinding is that the dynamic capacity is essentially the same as theequilibrium or static capacity because there is no mass transferresistance provided by the structure of the media to absorption.Equilibrium or static capacity refers to the quantity of the targetmolecule that is absorbed or adsorbed after a contact time sufficient toensure thermodynamically complete utility of absorption or adsorptionsites in the media. Unfortunately, because the surface area dictates thebinding capacity, there are limits to the binding capacity one canachieve for a given permeability due to the coupled flow and bindingproperties.

One example of a surface functionalized monolith is taught by Cerro etal., Biotechnol. Prog 2003, 19 921-927 (Use of ceramic monoliths asstationary phase in affinity chromatography), in which thin,surface-active only, agarose coatings on ceramic monoliths were createdby impregnating the monolith with the traditional hot solution ofagarose, followed by removal of excess hot agarose solution from thecells within the monolith using compressed air and subsequently coolingthe monolith to gel the agarose coating.

One of the major problems with this coating process is that the coatingsare difficult to effect on porous materials. In the article mentionedabove, the agarose had to be applied in a heated state (thus requiring asubstrate that is heat stable) making its application difficult tocontrol as gelling occurred as the temperature dropped. A furtherproblem is that only very thin coatings that have only surface activitycan be created as occurs in membrane adsorbers. In part, this may be dueto the method used for removing excess agarose. It may also be afunction of the agarose gel point and the higher viscosity that occursas the temperature of the agarose approaches the gel point. Moreover,the prior art process would be very difficult if not impossible withsubstrates having pores that are relatively small in comparison to thecell size of the monoliths of the prior art. The reason for thesedifficulties is that in some cases, air cannot be readily forced throughcertain porous materials without disrupting or otherwise damaging theporous structure, as is the case with certain fabrics or porousmembranes. Therefore relatively large pored, rigid monolithic structuresmust be used.

WO 00/44928 suggests another approach by forming a temperature stableagarose solution through the use of high levels (8 M) of chaotropes suchas urea. Agarose of this invention is imbibed into a porous support toform a continuous phase. Water is carefully added such that a gel layerforms at the interfaces between the agarose solution and the addedwater. The gel layer prevents migration of the agarose but allowsfurther migration of the water and urea molecules out of the agarosesolution into the added water. This process continues until the agarosesolution turns into a gel within the interstices of the pores of theporous substrate.

One major problem with this prior art method is that the process bywhich it is made causes the pores of the substrate to be substantiallyblocked, severely limiting convective flow through the porous support.Additionally, the diffusional resistance is high, limiting the abilityof the media to work rapidly.

What is desired is a porous adsorptive or chromatographic media havinggood convective and diffusional flow. More particularly, what is desiredis has a porous adsorptive or chromatographic media formed of a poroussubstrate having a porous coating that allows for good convective flowthrough the porous substrate with diffusive flow within the coatingitself that provides for good dynamic capacity.

SUMMARY OF THE INVENTION

The present invention relates to porous adsorptive or chromatographicmedia and methods of making them. More particularly, it relates toporous adsorptive or chromatographic media having one or more porouspolymeric based coatings and which have good convective and diffusionalflow characteristics and high dynamic capacity.

In order to overcome the limitations associated with bead, membrane andmonolithic media, a new composite media was invented. This novelcomposite media can increase dynamic binding capacity independent of theconvective flow properties. This novel media is a porous substratematerial (e.g. a membrane, non-woven, monolith, fiber and/or otherporous materials) coated with a permeable, porous hydrogel layer ofsufficient thickness to increase binding capacity, but no so thick as toadd significant diffusional resistance. The average thickness of thisadsorbing layer will vary depending on the porous materialcharacteristics and application targeted. The pores of the substrateafter coating preferably are permeable so as to allow cells and/orcellular debris to pass through them and the hydrogel layer must bepermeable to large entities such as proteins, DNA fragments and otherselected molecules.

The novel composite media also circumvents a problem associated withbead media. Typically, a bead media's mechanical strength is coupled tothe adsorbing material being used. For example, agarose beads are commonadsorbents in chromatographic applications. Agarose beads must bemodified, such as by crosslinking, to increase their mechanicalintegrity in order to operate at reasonable flow rates. Thismodification can change the binding performance of the resultingmaterial. Having the mechanical and binding properties coupled is alimitation associated with many bead media. The present invention reliesupon the underlying substrate for its strength and the coating for itsadsorptive properties.

Another advantage of this composite media is the ease of handling. Ingeneral, beads must be packed into a column. The quality of this packingdetermines the performance of the adsorbing bed. This adds anothersource of variability to the chromatographic process and must bevalidated before use. The novel composite media could operate in severaldevice forms, all which can be “packed” and validated prior to use,eliminating a source of variability during the chromatographic process.

Using the present invention, one can coat the surfaces of any article,including irregular materials such as porous materials, including theinterior surfaces of their pores, with a layer of the porous polymerwithout substantially blocking the pores of the substrate with thecoating so as to allow for convective flow through the porous substrate.Additionally, the coating is thick enough and porous enough to allow fordiffusive flow to occur within that layer itself.

In a preferred embodiment, the coating is in the form of a porouspolymer and is coated from a solution onto the surfaces of a preformedporous substrate. In one embodiment, all or a portion of the solvent inthe coating solution is evaporated before the coating material isgelled. In this embodiment, the optional use of wetting agents such assurfactants helps in forming relatively uniform and continuous coatings.

It is an object of the present invention to provide a media forchromatographic or adsorption separations comprising a porous coatedsubstrate the substrate being a porous (PS1), self-supporting structureand having one or more porous (PS2) coatings on at least a portion ofall surfaces of the substrate, the one or more coatings occupying afractional porosity of the structure of at least 1%, preferably fromabout 20% to about 65% and the one or more coatings being sufficientlyporous so as to allow for the adsorption of molecules such asbiomolecules within its bulk. The porous nature of the substrate is thatof large pores through which the mobile phase flows and contacts thestationary phase, that is, the media. This is referred to as “PorousSystem 1.” (PS1) The porous nature of the coating, Porous System 2,(PS2) refers to the pores in the coating, into which the dissolvedspecies of the mobile phase permeate, and have their velocity changed byinteraction with the coating or are adsorbed.

It is an object of the present invention to provide a media forchromatographic or adsorption separations comprising a porous coatedsubstrate the substrate being a porous (PS1), self-supporting structureand having one or more porous (PS2) coatings on at least a portion ofall surfaces of the substrate, the substrate being selected from thegroup consisting of fibers, woven fabrics, non-woven fabrics, felts,mats, monoliths and porous membranes, and one or more porous polymericcoatings on all surfaces of the substrate, the one or more coatingsoccupying from about 1 to about 50%, preferably from about 1 to about50% of the void volume of the pores of the substrate and the substratehaving good convective flow and diffusional flow.

It is an object of the present invention to provide a media forchromatographic or adsorption separations comprising a porous coatedsubstrate the substrate being a porous (PS1), self-supporting structureand having one or more porous (PS2) coatings on at least a portion ofall surfaces of the substrate, the substrate selected from the groupconsisting of fibers, woven fabrics, non-woven fabrics, felts, mats,monoliths and porous membranes, and one or more porous polymericcoatings on all surfaces of the substrate, the one or more coatingsoccupying from about 10 to about 50% of the void volume of the pores ofthe substrate and the media has good convective flow and diffusionalflow.

It is an object of the present invention to provide a media forchromatographic or adsorption separations comprising a porous coatedsubstrate the substrate being a porous (PS1), self-supporting structureand having one or more porous polymeric (PS2) coatings on at least aportion of all surfaces of the substrate, the one or more coatingsoccupying from about 10 to about 50% of the void volume of the pores ofthe substrate and dynamic binding capacity of the media is independentof the convective flow properties of the media.

It is an object of the present invention to provide a media forchromatographic or adsorption separations comprising a porous coatedsubstrate, the substrate being a porous (PSI), self-supporting structureand having one or more porous polymeric coatings (PS2) on at least aportion of all surfaces of the substrate, the base substrate being aporous, self-supporting structure selected from the group consisting ofwoven fabrics, non-woven fabrics, felts, mats, fibers, monoliths andporous membranes, and one or more porous coatings on all surfaces of thesubstrate, the one or more coatings occupying from about 10 to about 50%of the void volume of the pores of the substrate, the one or morecoatings being formed of one or more polymers and the substrate havinggood convective flow and diffusional flow.

These and other objects of the present invention will be described ingreater detail below in the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a planar view of a portion of an embodiment according tothe present invention.

FIG. 2 shows a cross sectional view of a portion of an embodimentaccording to the present invention.

FIG. 3 shows a cross sectional view of a device containing the media ofan embodiment according to the present invention.

FIG. 4 shows a cross sectional view of a second device containing themedia of an embodiment according to the present invention.

FIG. 5 shows a cross sectional view of another device containing themedia of an embodiment according to the present invention.

FIG. 6 shows a cross sectional view of a further device containing themedia of an embodiment according to the present invention.

FIG. 7 shows a cross sectional view of an additional device containingthe media of an embodiment according to the present invention.

FIG. 8 shows a cross sectional view of another device containing themedia of an embodiment according to the present invention.

FIG. 9 shows a planar view of another embodiment of the media accordingto the present invention.

FIG. 10 shows a planar view of another embodiment of the media accordingto the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a porous chromatographic or adsorptivemedia having a porous, polymeric coating (PS2) formed on a porousself-supportive substrate (PS1) such that the coating is at least 10% ofthe void volume of the structure and is capable of adsorbingbiomolecules, such as proteins, DNA fragments or other selectedmolecules within its bulk. More particularly, the present inventionrelates to a porous chromatographic or adsorptive media having a porouspolymeric coating (PS2) formed on a porous self-supportive substrate(PS1) such that the media retains at least 20% of the initial voidvolume of the substrate and has good convective and diffusional flow andhigh dynamic capacity.

FIG. 1 shows a first embodiment of the present invention. The media isformed of a porous substrate (PS1) 2, the surfaces of which are at leastpartially coated by one or more porous coatings (PS2) 4. As can be seenfrom the Figure, the substrate has a series of large pores 6 separatedfrom each other by interconnecting walls 8.

FIG. 2 shows a close up cross-sectional view of one pore 6 of thesubstrate 2 with the coating 4 in place. As shown, the coating is atleast partially in contact with the surface of the substrate; preferablyit is substantially in contact with surface of the substrate to form arelatively uniform coating surface.

Such a substrate can be a fiber, non-woven fabric, woven fabric, mat,felt, membrane or a monolith as explained in further detail below. Thesubstrate, even with the coating(s), has high permeability and good flowand capacity characteristics. The substrate is self-supportive andprovides a platform or structural network for the coating(s).

It is preferred that the substrate selected be highly porous, so thatthere is minimal, but sufficient wall or solid material within it toprovide the structural integrity and high porosity and flow. The poresizes of pore system may vary from about 1 to about 1000 microns,preferably 10 to 300 microns, more preferably from about 50 to about 200microns and more preferably from 50 to 100 microns, depending upon thefluid and the constituent that is desired to be captured from it. Forexample, in an application to capture a desired protein from anunclarified, lysed cell broth, the pores of the substrate should besufficiently large enough to allow good permeability at high flow ratesof the broth through the media, i.e., the coated substrate, while stillallowing for a high level of capture on a single pass, such as greaterthan 50%. In the above application, a pore size of from about 100 toabout 300 microns would be preferred. In an application starting withclarified or clean feedstreams, the pore size can be smaller, form about30 to about 60 microns. For laboratory devices such as syringe filters,or microtiter plates, which are used with a variety of solutionconditions, smaller pores are preferred when clean, very dilutesolutions are used. These pores are from about 0.1μ to about 10 μ.

The coating(s) themselves are also porous in nature (PS2) so that theyare permeable to biomolecules. Preferably they are capable of adsorbingbiomolecules within their bulk, namely within the pores formed withinthe coating(s). The coating(s) are thick enough to create these poresand have some diffusional flow into them, thereby increasing overallcapacity of the structure above that of the surface alone and in someapplications selectivity of the capture, but they are sufficiently thinso that the diffusion length is limited and not a negative factor inperformance either in capturing or releasing the biomolecules.

The coating(s) typically constitute at least 1% of the total volume ofthe coated substrate. Preferably they are from about 5% to about 80% ofthe total volume of the coated substrate.

By another measure, on average, the coatings reduce the average diameterof the substrate pores by an amount from about 1% to about 80%,preferably from about 10% to about 50%, more preferably from about 20%to about 50% from that of the uncoated substrate.

By another measure, the coatings reduce the permeability of thesubstrate by an amount from about 5% to about 80% of that of theuncoated substrate.

Another method for determining the amount of coatings used is fractionalporosity, which is important for ensuring the flow through the coatedsubstrate. Fractional porosity is the ratio of volume within the coatedsubstrate that is available to the solution being processed to the totalvolume of the coated substrate. A higher fractional porosity gives ahigher inherent flow capacity to the coated substrate. For the coatedsubstrates of the present invention, preferred fractional porosities arefrom about 0.35 to about 0.55.

The coating(s) are generally from about 1 to 100 microns in thickness,preferably from about 2 to about 20 microns in thickness and morepreferably from about 5 to about 15 microns in thickness. Thicknessrefers to the change in the characteristic measure of the solid phase ofthe substrate. For example, for a woven or non-woven fabric, the changein the radius of the characteristic fiber is the coating thickness. Thediameters of pores of the coating(s) (PS2), may vary within the range ofthose commonly used in chromatography or from about 1 to about 200nanometers, preferably from about 1 to about 100 nanometers, morepreferably from about 1-50 nanometers. They should be of a sizesufficient to allow for the passage or permeation of the desiredmaterial into them, such as proteins, DNA or RNA fragments, plasmids orother biomolecules, synthetic molecules such as oligonucleotides, otherselected molecules and the like.

In a preferred embodiment the coating covers the surfaces of thesubstrate to a substantially uniform thickness. To accomplish thisrequires routine trials in which the coating solution viscosity,substrate pore size, method of removing excess solution and dryingprocedures are optimized to obtain this end. In general, a practitioner,once aware of the teachings of this invention will determine anapproximate coating thickness that will optimize capacity and adsorptionand release rates for the desired selected molecule. He will then choosea substrate with a pore size and a porosity such that this thicknesswill not overly reduce flow. Routine trial and error experimentation,based on the teachings of the present invention, will provide a skilledpractitioner a route to the correct formulation and drying method.

In a preferred embodiment, substantially all surfaces are covered withthe porous coating, preferably of a uniform thickness.

The structure of the present invention has good hydraulic permeability.Hydraulic permeability is the measure of flow through the media, givenas volume flow per facial or frontal area per time, normalized forpressure. Flow is the volume passing through the media per unit time.The present invention has inherent flow even at relatively low pressure(1 bar), and has a stable flow at relatively high flow rates such as 300cm/hr or greater. Preferably, flow is relatively linear with pressurefrom about 1 cm/hr to about 500 cm/hr.

The media also has good capacity. Generally, this means there is arelatively high surface area available to be in direct contact with thefluid flowing through the structure as compared to the surface area ofthe underlying substrate. Typically, a media according to the presentinvention has a surface area that is at least 25%, preferably, 50% andpreferably 75% higher than the surface area of the substrate itself dueto the porosity of the coating.

The substrate may be a fiber, a sheet such as a woven fabric, anon-woven, a mat, a felt or a membrane or it may be a three dimensionalstructure such as a sponge, poly(HIPES) or other monolithic structuresuch as a honeycomb, or a porous bead such as,a controlled pore glass,porous styrene beads, silica, zirconia and the like. Preferably, thesubstrate is a sheet formed of a woven or non-woven fabric or amembrane.

Fibers may be of any length, diameter and may be hollow or solid. Theyare not bonded together as a substrate (although as discussed below,they may be formed into an unitary structure after application of thecoating) but are individual discrete entities. They may be in the formof a continuous length such as thread or monofilament of indeterminatelength or they may be formed into shorter individual fibers made bychopping fibrous materials such as non-woven or woven fabrics, cuttingthe continuous length fiber into individual pieces, formed by acrystalline growth method and the like.

Non-woven fabrics are flat, porous sheets made directly from separatefibers bonded together by entangling fiber or filaments, thermally orchemically. Typically, nonwoven fabric manufacturers supply media havingfrom 1 to 500 micron mean flow pore (MFP) ratings. For non-wovenfabrics, the porous structure is the entangled fibers, and porosityrefers to the tortuous spaces between and among the fibers. Porosity hasa similar meaning for felted fabrics. A preferred non-woven is byFreudenberg Nonwovens NA of Lowell, Mass. and is type FO2463.

Woven fabrics are produced by the interlacing of warp fibers and weftfibers in a regular pattern or weave style that is at some predefinedangle to each other. Typically the weft is at an angle of about 90degrees to that of the warp. Other commonly used angles include but arenot limited to 30, 45, 60 and 75 degrees. The fabric's integrity ismaintained by the mechanical interlocking of the fibers cause by theweaving process. Drape (the ability of a fabric to conform to a complexsurface), surface smoothness and stability of a fabric are controlledprimarily by the weave style, such as plain, twill, satin, basket weave,leno, etc. In this case, the substrate porosity is the space between thefibers and is of a less tortuous nature.

Monoliths are blocks of porous material. They can be rectangular,cylindrical, or foamed into other shapes. Examples are ceramicmonoliths, which are ordered structures of packed rectangular ortriangular capillaries. These are supplied by Engelhard, Inc ofHuntsville, Ala. and Corning, Inc of Corning, N.Y. One form of polymericmonoliths are made from sintered plastic particles by Porex Corporationof Fairbum, Ga.

Poly(HIPES) [high internal phase emulsion] materials are mechanicallystable isotropic, open celled polymeric foams. These, and othermacroporous polymer structures are described in “Porous polymers andresins for biotechnological and biomedical applications” H.-P. Hentzeand M. Antonietti Reviews in Molecular Biotechnology 90 (2002) 27-53

The substrate may be formed from a variety of materials including glass,plastics, ceramics and metals.

Borosilicate glass is one example of a suitable glass. It can be formedas fibers, glass mats or porous beads such as the controlled pore glassbeads available from Millipore Corporation of Billerica, Mass.

Various ceramics based on the more conventional silicate chemistries ormore exotic chemistries such as yttrium, zirconia, titanium and the likeand blends thereof can be used. They can be formed into beads, fibers,mats, felts, monoliths or membranes.

Metals such as stainless steel, nickel, copper, iron or other magneticmetals and alloys, palladium, tungsten, platinum, and the like maybemade into various forms including fibers, sintered sheets andstructures, such as sintered stainless steel or nickel filters, wovenscreens and non-woven mats, fabrics and felts such as stainless steelwool.

The preferred substrate is made from plastic, more preferablythermoplastics. Preferred thermoplastics include but are not limited topolyolefins such as polyethylenes, including ultrahigh molecular weightpolyethylenes, polypropylenes, sheathed polyethylene/polypropylenefibers, PVDF, polysulfone, polyethersulfones, polyarylsulphones,polyphenylsulfones, polyvinyl chlorides, polyesters such as polyethyleneterephthalate, polybutylene terephthalate and the like, polyamides,acrylates such as polymethylmethacrylate, styrenic polymers and mixturesof the above. Other preferred synthetic materials include celluloses,epoxies, urethanes and the like.

Suitable coating materials include but are not limited to the followingpolymers, polyvinyl alcohols, acrylates and methacrylates,polyallylamines, polysaccharides such as agaroses, dextrans,cyclodextrans, celluloses, substituted celluloses and the like and maybe used in concentrations from about 0.5 to about 20, preferably fromabout 1 to about 10% by weight of the solution.

A preferred process for forming the present invention comprises thesteps of a) forming a room temperature stable coating solution of one ormore polymers, a solvent for the polymer(s) and one or moregel-inhibiting agents, if required; b) wetting a porous substrate withthat solution, optionally removing excess solution; c) evaporating thesolvent from the solution to cause the coating to conform to thesurfaces of the substrate; d) if necessary due to use of gel-inhibitingagents, welting the coated substrate with a gelling agent that is anonsolvent for the polymeric and is a solvent for the gel-inhibitingagents; and e) rinsing the coated substrate. Additionally, optionalsteps include crosslinking the substrate and/or adding a functionalityto the surface of the coating.

For those polymers that are either soluble in water at room temperature,such as some dextrans and low gel point agaroses or which are notsoluble in water, such as cellulose, polyvinyl alcohols, other vinylcontaining compounds, acrylates, methacrylates and the like, otherprocesses and solvents can be used.

For example, for cellulose, one may use a solvent such asN,N-dimethylacetamide and saturate the solvent with a salt such aslithium chloride and coat the material on to a substrate and then removethe solvent and salt in water.

For dextrans and low gel point agaroses that are soluble in roomtemperature water, the use of the gel-inhibiting agent is not required.A preferred method for these materials is to form a coating dissolved inwater with a suitable crosslinker such as polyethylene glycol diglycidylether, one or more porogens such as polyacrylamide solution (10% inwater). The coating is applied, dried and then the media is heated tocrosslink the dextran or low gel point agarose. The porogen is removedby washing or extraction in water.

Homogeneous water solutions of polyvinyl alcohol (PVA) and polyalkyleneglycols can be used to make porous coatings. PVA polymers useful forthis purpose are those with an average degree of polymerization in therange of 500 to 3,500 and a degree of saponification in the range of 85to 100 mole percent, or PVA copolymers containing less than 10 molepercent of monomers such as ethylene, vinyl pyrrolidone, vinyl chloride,methyl methacrylate, acrylonitrile and/or itaconic acid. Polyalkyleneglycols employable according to this invention have an average molecularweight in the range of 400 to 4,000 and, preferably, in the range of 600to 3,000 and have a carbon-to-oxygen ratio of not more than 3.

A typical solution comprises polyvinyl alcohol polymer containing 15 to150 weight parts of a polyalkylene glycol per 100 weight parts ofpolyvinyl alcohol. PVA polymer is coagulated by one of the followingprocedures: (1) extruding an aqueous solution of PVA polymer into anaqueous solution of a dehydrating salt such as sodium sulfate, ammoniumsulfate, potassium sulfate or sodium phosphate; (2) extruding an aqueoussolution of PVA polymer into an aqueous solution of alkali, e.g., sodiumhydroxide potassium hydroxide and lithium hydroxide, and (3) extrudingan aqueous solution of PVA polymer containing boric acid or a saltthereof into an aqueous alkaline solution of sodium hydroxide and sodiumsulfate.

PVA solutions having an upper critical solution temperature (UCST) canbe used to make porous PVA coatings. Water is a preferred solvent, butother solvents can be used, including alcohol/water mixtures, dimethylsulfoxide, dimethylformamide, dimethylacetamide andN-methylpyrrrolidone. These solutions are homogeneous above their UCSTand must be coated at a temperature greater than their UCST. Solutionswith UCST between about 30° C. to about 95° C. can preferably be used.These solutions can be made from PVA solutions containing polyethyleneglycol, polypropylene glycol, tetraethylene glycol, triethylene glycol,ethylene glycol, lower alkyl alcohols, polyhydric alcohols such asglycerine and butanediol, and lower alkyl esters, such as ethyl lactateor butyl lactate. Thickening additives such as boric acid or acetic acidor mixtures can be used in small quantities, less than 1%. PVAconcentrations of 5-20% are preferred. The amount of additive isdetermined experimentally to produce a solution with the desired UCST.The amount of additive depends on the amount and molecular weight of PVAand selection of additive.

The coated substrate can be made by a variety of methods. The coatingcan be formed from a preformed polymer. The general method is to contactthe substrate with a solution of the polymer, and to remove excesssolution. The next step is to fix the polymer to the substrate. This isdone by removing the solvent and any additives that were in thesolution. The polymer can be crosslinked, either during these steps, orin a separate step.

For simple solutions, all or a portion of the solvent may be removed byevaporation. Or the solution may contain a volatile solvent as part ofthe composition of the mixture making up the solvent. Then it may proveto be beneficial to remove the volatile solvent and/or a portion of themain solvent by evaporation with or without heating. The solvent andadditives can also be removed by immersion in a liquid that does notdissolve the polymer, or remove any reactants (see below). For polymersthat are insoluble in water, removal of solvent and additives thatenhance solubility leaves the polymer permanently affixed to thesubstrate. There is usually a final washing and rinsing procedure. Thefixed polymer can be then dried, or kept in a wetted state.

The polymer coating can be crosslinked either during the steps above orin a separate step after drying or after rinsing. These methods will bedescribed in the further in this section and in the Examples.

In another method of forming a porous hydrogel on the substrate, asolution of reactive monomers is contacted with the substrate, and/or aninsoluble or crosslinked polymer is formed in situ. The method hassimilarities with the preformed polymer method just described.

The solution can have multiple components. There will be a solvent, madeof one or more solvents and possibly other components, such asnon-solvents or poor solvents. There will be one or more monomers. Thewill usually be one or more multifunction molecules for crosslinkingpurposes. There will be in most cases a polymerization initiator. Theremay be a non-reactive or in some cases, a reactive polymer, forviscosity increasing purposes. There may be a surfactant to improvecoatability of the solution to the substrate.

The solvent is usually aqueous, which includes 100% water, and mixturesof water and water miscible organic solvents to increase solubility ofthe monomers, crosslinkers, polymers, etc. Alcohols of less than fourcarbons, ketones such as acetone, MIBK, are typical. There may be caseswhere solvents such as dimethyl acetamide, dimethylsulfoxide, N-methylpyrrolidone, tetrahydrofuran may be needed. It is of course possible tomake hydrogel polymers out of organic solutions if the final product isdried and rehydrated before use.

Monomers used to make ionically uncharged hydrogels usually containhydroxyl or amide groups. Examples of hydrophilic monomers are2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate,hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate,methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate,poly(ethylene glycol)methacrylate, methoxy-poly(ethyleneglycol)methacrylate, methacrylic acid, sodium methacrylate, glycerolmethacrylate, hydroxypropyl methacrylate, and hydroxybutylmethacrylates, acrylamide monomers such as acrylamide and substitutedacrylamines, e.g., dimethylacrylamide, diethylacrylamide, diacetoneacrylamide, N-(3-aminopropyl)methacrylamide, and analogousmethacrylamide monomers, N-vinyl-pyrrolidines and diacroylpiperazine.

Charged hydrogels can be made from acrylic acid monomer,2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 3-sulfopropylacrylate potassium salt , 2-(acryloyloxy)ethyltrimethyl-ammonium methylsulfate, 4-vinylpyridine, acrylic acid, methacrylic acid,(3-(methacryloylamino)propyl)trimethylammonium chloride,(3-acrylamidopropyl)trimethylammonium chloride andaminopropylmethacrylamide.

Crosslinkers which can be used include ethylene glycol dimethacrylate,diethylene glycol dimethacrylate, triethylene glycol dimethacrylate andpoly(ethylene glycol) dimethacrylate.oligo(polyhydroxylalkyl)silylmethacrylate ethylene-bis-acrylamide,methylene-bis-acrylamide, and piperazine diacrylamide.

Viscosity enhancing polymers useful for aqueous solutions includepoly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-copoly(propyleneoxide) block copolymers, and polysaccharides.

Examples of suitable initiators include, for example, ammoniumpersulfate, potassium persulfate, azobis(4-cyanovaleric acid, Irgacure2959 (Ciba-Geigy, Hawthorn, N.Y.),2,2′-azobis(2-amidino-propane)hydrochloride and the like., potassiumpersulfate, 2,2′-azobis(2-amidinopropane)hydrochloride, potassiumhydrogen persulfate.

In the method described below, the ranges of components are as follows(all concentrations are in weight %);

Monomer concentrations are from about 2% to about 20%, with a preferredrange of from about 5% to about 15%.

Crosslink concentrations are from about 1% to about 10%, with apreferred range of from about 3% to about 6%.

The polymer additive concentration is from about 2% to about 30%, with apreferred range of from about 5% to about 20%. The preferred polymersare low molecular weight polyethylene glycols, of about 8000 molecularweight. High molecular weight polymers tend to be difficult to saturateporous substrates and are difficult to remove in the final washingsteps.

Initiator concentrations are from about 0.1% to about 10%, with apreferred range of from about 0.25% to about 5%.

Surfactant concentration is from about 0.01% to about 10%, with apreferred range of from about 0.05% to about 5%. Anionic surfactants,such as sodium lauryl sulfate are a preferred class of surfactants.

The general method is to make up a solution of the monomer(s) andcrosslinker, with the other components, in a water organic solventmixture. The organic solvent is usually a volatile solvent that will beeasily evaporated. Methanol and acetone are examples of preferredorganic solvents. The volatile component can make up are from about 10%to about 50% of the total solution, with a preferred range of from about20% to about 40%. In a preferred embodiment, the final composition issuch that the viscosity allows easy penetration of the solution into thesubstrate, and complete wetting of all surfaces.

The substrate is contacted and saturated with the solution, and excesssolution removed by rollers, scraping, or other means. The solution inthe welted substrate is allowed to evaporate, generally at lowtemperatures, to remove the volatile organic component. This decreasesthe overall mass in the substrate and increases the concentration of thedissolved material in the solution and concurrently, the viscosity. Theresult is that the remaining solution pulls back the solid phase of thesubstrate, e.g., the fibers in a non-woven fabric, and leaves a regionin the substrate with no solution, a convective pore. It is also animportant aspect of the process that some amount of water remain toallow the polymerization reaction to proceed.

One can form coatings from monomeric as well as polymeric systems. Forexample one can make acrylate, acrylamide or allylamine porous coatingsfrom a monomeric system and polymerize them as part of the process. Onetakes the selected monomer or monomers, polymerizing agent, porogen andsolvents and/or diluents and applies the resultant solution to a poroussubstrate. The solution is dried to remove at least a portion of thesolvent and/or diluent and it is then polymerized such as by UVradiation. The porogen is then removed and the coated media is formed

The coating solution of the above preferred process is now describedwith reference to being formed of polysaccharide, such as agarose, oneor more gel-inhibiting agents such as various salts, and one or moresolvents such as water for the coating material.

The polysaccharide, one or more gel-inhibiting agents and solvent aremixed and heated above the melting point of the polysaccharide. Themelting point varies for different grades of polysaccharide, buttypically for agarose it is between about 90° C. and 98° C., mostcommonly between 92° C. and about 98° C. This may be done in one step bycombining and heating all three components together. Alternatively andpreferably, one can first add the polysaccharide in powdered form to asolvent such as water and disperse the powder into a slurry. It is thenheated to dissolve the polysaccharide and cooled it to form a gel. Thegel is then reheated to a liquid solution and the gel-inhibiting agentis added and dissolved into the solution. Once it has completelydissolved, the solution is cooled, typically to about room temperature(23° C.).

In either method, the polysaccharide is dissolved by heating thedispersion in a range of from approximately 95° C. to the boilingtemperature. This can be done, for example, in a stirred vessel, or in amicrowave oven. The hot solution may be filtered if needed to remove gelor other particles. Once a clear solution is formed, the solutionpreferably is allowed to cool.

One may allow this cooling to occur naturally or one may, if desired,affirmatively cool the solution. At room temperature, the solution is astable, non-gelled solution. The gel point (typically between 30° C. and68° C.) is suppressed by the addition of the one or more gel-inhibitingagents.

The type of polysaccharide used will be determined by the propertiesdesired of the final coating. The dispersion is made so that the finalconcentration of polysaccharide is between about 0.1% to about 20%,preferable between about 1% to about 10%, more preferably between about2% to about 6%, by weight of total final solution.

While water is the preferred solvent for the polysaccharide, a minoramount, up to 20% by weight of the dissolving solution, of co-solventmay be added to improve solubility of the polysaccharide. Examples ofsuitable co-solvents are dimethylacetamide or dimethylsulfoxide. Othersare known to those skilled in the art.

A gel-inhibiting agent is used to prevent the gel from re-gelling aftermelting and cooling. The agent may be added to the hot solution, or tothe solution after cooling to a temperature above the gel point, or atany time prior to complete gelation. In a preferred method, agel-inhibiting agent is added to the gelled solution. When added to thegel, the heat of solution tends to assist dissolution of the agent.Preferred agents are based on zinc, lithium or sodium salts such asZnCl₂, LiCl, and NaOH. Zinc salts can be used at a concentration ofgreater than about 15% by weight, based on the dissolving solution, upto the solubility limit, about 45.8% for ZnCl₂, and about 54.6% forZn(NO₃)₂. Lithium salts can be used at concentrations greater than about18%, to their solubility limit, about 45.8% for LiCl, 51.0% for LiNO3,or 54.0% for LiSCN. NaOH can also be used at about IM concentration. Apreferred salt is ZnCl₂.

The gel-inhibiting agent may also be a chaotrope, a small solute thatenhances the ability of the solvent to dissolve polysaccharides.Non-limiting examples of such gel-inhibiting agents are urea andguanidinium salts at concentrations up to 8M, inorganic salts andbuffers such as KI, NaI, MgCl₂, potassium dihydrogen phosphate, disodiumhydrogen phosphate, tris(hydroxymethyl)aminomethane, sodium tetraborate,and others known to those skilled in the art.

A porous substrate is then chosen from any of those discussed above.

The room temperature stable solution can be used as is for coating. Itis preferable to add gel-modifying materials to the solution in order tomodify and control the structure and properties of the final coating.

One class of added gel modifying materials comprises volatile organics,miscible with the solution. Examples are monohydric alcohols such asmethanol, ethanol, and propanols. These can be used up to concentrationsthat give a slightly cloudy solution. Higher amounts of these alcoholscan cause precipitation of the agarose. Preferred amounts areequi-volumetric with the water in the solution, more preferred is to addthe alcohols to about 40% to about 60% of the water. A preferred alcoholis methanol. Miscible ketones such as acetone can also be used, but caremust be used as the solubility of agarose is less in ketone-watermixtures. Any mixture of two or more of these materials is alsocontemplated.

Another class of added gel modifying materials comprises non-volatilemiscible organics. Non-limiting examples of these included glycerine,ethylene glycol, methyl pentane diol, diethylene glycol, propyleneglycol, triethylene glycol, the methyl, ethyl, or n-butyl ethers ofethylene glycol, the dimethyl or diethyl ethers of ethylene glycol,ethylene glycol dimethyl ether acetate ethylene glycol diethyl etheracetate, diethylene glycol methyl ether, diethylene glycol ethyl ether,diethylene glycol n-butyl ether, diethylene glycol dimethyl ether,diethylene glycol diethyl ether, diethylene glycol dimethyl etheracetate, diethylene glycol diethyl ether acetate, N-methyl morpholine,N-ethyl morpholine, and the like. polyethylene glycols of low molecularweight are also examples of materials that are in this class. Anymixture of two or more of these materials is also contemplated.

Another class of added gel modifying materials comprises water-solublepolymers, which include by way of examples, polyvinyl pyrrolidone,polyvinyl alcohol, polyethylene glycols, dextrans, and water-solublepolyacylamides, including substituted polyacylamides, such aspolydimethylacrylamide. These polymers are believed to act as“porogens.” That is, they control the amount of volume of the coatingthat is freely permeable to dissolved solutes when the coated poroussubstrate is in use.

These polymeric additives can be used as blends with the agarose in theinitial dissolution step, or they can be dissolved in the solution withor after the added materials just discussed are mixed. Care must betaken not to add an excessive amount of polymer, as coagulation of thesolution may occur. Ratios of polymer to agarose of from about 0.1 to 10are possible. Preferred polymers are polyvinyl alcohol and dextrans.Polyacrylamides have also been found to be useful.

To obtain optimum coatability of the solution, one or more surfactantsare added to the solution. Each combination of solution type andsubstrate may require some experimentation to determine the optimum typeof surfactant for the desired coating system. Anionic surfactants havebeen found to be useful, with anionic fluorosurfactants being preferred.Of these, 3M FC-99 and FC-95 or equivalents from other suppliers aremost preferred. These can be used in concentrations of from about 0.001%to about 10%, preferably from about 0.01% to about 5%,

The substrate is impregnated with the coating such as soaking thesubstrate in a bath of the coating, applying the coating material by adoctor blade, spray nozzle, curtain coater, roll coater, extrusioncoater or any other method known to one of ordinary skill in the art toapply a coating to a porous substrate. Excess coating material isremoved such as by blotting or shaking the coated substrate, squeezingsuch as through a nip roller, scraping the surface of the coatedsubstrate or by blowing air or a gas at the substrate's surface.

The solvent for the coating is then at least partially removed byevaporation. Preferably, this is a controlled evaporation such that thecoating evaporates relatively uniformly throughout the entire substrate.The use of heat warmed air (preferably between 20 and 80° C.), microwavedrying, vacuum oven drying and the like to control and/or spedevaporation may be used if desired. This causes a polysaccharide coatingto be formed on the substrate surfaces that is dry to the touch, butstill contains some residual moisture within it.

The coated substrate is then subjected to a gelling agent that removesthe salts from the coating and causes the polysaccharide to form aporous hydrogel coating. The agent can be water, if done so as not tooverly swell the coating. This can be done by controlling the previoussolvent removal/drying step so that the water extracts thegel-inhibiting agents before deleterious swelling can occur. Once alarge proportion of the gel-inhibiting agents are removed, swelling inwater is reduced to a minimum. The use of water with added salts reducesthe tendency of the aqueous rinse to swell the coating.

The use of organic solvents as the gelling agents to remove thegel-inhibiting agents without swelling the coating is preferred.Acetone, methanol, ethanol, or propanols are useful. Mixtures of fromabout 25% to about 95% acetone or methanol in water have been found tobe useful. Similar acetone/methanol mixtures are also useful.

The substrate may be sprayed with the gelling agent, although preferablyit is immersed into a bath containing the agent. The agent is preferablyapplied at room temperature.

The coated substrate is then rinsed with water and maintained preferablyin a wet state. This rinsing step is generally done at temperaturesbetween about 15° C. and about 50° C., preferably between 20° C. and 50°C. The coated substrate will have at least a portion of all of itssurfaces (facial and interior surfaces) covered with a coating that ispermeable to biomolecules. Preferably the coating is relativelyuniformly applied to the substrate. More preferably, substantially allof the surfaces are covered by the coating. Also preferably, the coatingis of relatively uniform thickness throughout the substrate.

The coating may then be crosslinked if desired by any of the chemistriescommonly used in the industry to crosslink materials containing multiplehydroxyl groups, such as polysaccharide beads, these chemistries beingas non-limiting examples, epichlorohydrin or other multifunctional epoxycompounds, various bromyl chemistries or other multifunctional halides;formaldehyde, gluteraldehyde and other multifunctional aldehydes,bis(2-hydroxy ethyl)sulfone, dimethyldichloro-silane, dimethylolurea,dimethylol ethylene urea, diisocyanates or polyisocyanates and the like.

For dextran coatings, the use of a crosslinking step is required.Typically this occurs after drying of the coating to the substrate butbefore rinsing, although some partial crosslinking of the solutionbefore coating is applied may be done if desired.

It may also have one or more functionalities applied to it, includingligands, such as Protein A or Protein G, natural or recombinatorilyderived versions of either, modified versions of Protein A or G torender them more caustic stable and the like, various chemical ligandssuch as 2-aminobenzimidazole (ABI), aminomethylbenzimidazole (AMBI),mercaptoethylpyridine (MEP) or mercaptobenzimidazole (MBI), or variouschemistries that render the coating cationic, anionic, philic, phobic orcharged, as is well-known in the art of media formation.

Functional groups used in liquid chromatography that are adaptable tothe present invention include groups such as, but not limited to, ionexchange, bioaffinity, hydrophobic, groups useful for covalentchromatography, thiophilic interaction groups, chelate or chelating,groups having so called pi-pi interactions with target compounds,hydrogen bonding, hydrophilic, etc.

The media can then be placed into a holder and have a liquid stream(containing one or more desirable components capture in it) run throughthe media so that the desired components are separated from the rest ofthe liquid. Typically, it is the desired components that are capturedfrom the liquid and the rest of the liquid including impurities passesthrough. Alternatively, the desired components may pass through andimpurities can be captured by the media. The composite is washed toremove any unbound materials and then the captured material is elutedusing a change in ionic strength, pH or the like.

If desired or required, one may apply a second or even more coatinglayers to the first in order to reach the desired thickness ofcoating(s), to change their chemical nature (i.e., layers of differentcoatings) and the like.

In one form, a series of individual fibers may be placed into acontainer having a porous surface such as a frit or filter and beretained there by that surface. Such devoices include but are notlimited MICROCON® centrifugal filter devices available from MilliporeCorporation of Billerica, Mass., STERICUP® filtration devices availablefrom Millipore Corporation of Billerica or closed test tube such as isavailable from Fisher Scientific and the like. Fluid is added on top ofthe series of fibers and the selected molecule is adsorbed by the media.In using filter devices, the fluid is filtered through the poroussupport and the fibers are then washed and then treated with an eluantto recover the selected molecule. In a closed system the fluid after asuitable residence time can centrifuged and the liquid decanted. Asimilar process then occurs with the washing fluid and the eluant torecover the selected molecule of interest. Optionally, the series offibers can be laid together and then the coating can be crosslinkedbefore use to help forma unitary structure. Alternatively, the fibersafter coating can be bound to each other by other mechanisms such asheat, adhesives and the like provided they don't adversely affect theadsorptive properties of the coated fibers.

In another form the continuous coated fiber is first formed by forming acoating on a continuous fiber such as a thread or monofilament. This canbe done in a number of ways including running the fiber through a bathof coating, spraying the fiber with coating or running the fiber througha die coater.

The coated fiber is then wrapped or wound on to a mandrel that forms aninner porous core for a device. The wraps are spaced apart from eachother and at various angles to the wrap below and above it so thatconvective pores are formed throughout the structure. The fibers maybecrosslinked after they have been wound into the porous depth filter. Thewound core can be used as made and placed into a holder such as a filterholder, a chromatography column, or a capsule with flow distributorssuch as frits and the like at each end adjacent the inlet and outlet tothe device. Fluid is then flowed through the device axially and theselected molecule is captured. Alternatively, fluid can be introducedalong either the inner core or outer perimeter of the wound media andflow radially through the media exiting the opposing perimeter surface.

In applications where the binding affinity is large and flow uniformityis not a requirement, one can form a core having a wound, coated fiberor fibers on its outer surface. One of the two ends can be attached toan outlet and the other sealed, typically with an endcap. The core canbe placed in a filter housing or a disposable capsule having an inletsuch that fluid enters the housing or capsule through the inlet, flowsthe outer surface of the wound coated fibers and makes its way to thecore where it passes through and then leaves the housing/capsule throughthe outlet. The selected molecule is captured by the coating as thefluid passes through the wound structure.

In another form, a device incorporating the present media may be asimple plastic or metal filter holder such as a SWINNEX® plastic filterholder available from Millipore Corporation of Billerica, Mass. or astainless steel filter holder available from Millipore Corporation ofBillerica, Mass. These devices as shown in FIG. 3 are formed of twohalves 10 and 12 each having a port 14 and 16 respectively for liquidflow into or out of the device. In this case half 10 has inlet 14 andhalf 12 has outlet 16. One or more sheets of media 18 (in this exampleone sheet is shown) according to the present invention is placed betweenthe two halves 10 and 12 of the device and simply sealed between byclamping pressure, typically through a mated male/female threads 20 and22 as shown.

Another embodiment would permanently seal one or more layers between twoplastic filter holder halves as shown in FIG. 4. Here the first half 30has an inlet port 32 and the second half 34 has an outlet port 36. Theouter peripheral edge 38 of the substrate 40 is trapped between the twohalves 30 and 34. The outer peripheral edges 42 and 44 of the two halves30 and 34 respectively are sealed together by an overmold of plastic 46to form a liquid tightly sealed device. One such device is sold asMILLEX® filter available from Millipore Corporation of Billerica, Mass.

The media may also be incorporated into a filter cartridge device FIG. 5either as a pleated media or as a depth filter. A cartridge 50 containsa central core 52 that is connected to a first endcap 54 which forms anoutlet 56 from the cartridge 50. The media 55 is upstream from the core52. If desired a support layer or cage (not shown) may be used either onthe outside or inside or both sides of the media 55 as is well known inthe art. The media 55 is liquid tightly sealed to the first endcap 54and a second endcap 60 at its respective ends so that all fluid enteringthe cartridge 50 through inlet 53 must flow through the media 55 beforeentering the core 52. An outer sleeve 58 surrounds the media 55 and isalso sealed to the endcap 54 such as by mated threads, clamps,adhesives, solvent bonding, ultrasonic welding and the like. In thepleated form, one or more layers of media may be used. Likewise in thedepth filter form the media maybe one thick layer, such as a mat or afelt or a monolith or it may be a single sheet of media that has beenrolled up upon itself or it may be series of individual sheets on top ofeach other and sealed along their open two edges (not shown). Thecartridge may go into a reusable housing that is liquid tight or it mayhave the outer sleeve liquid tight (as shown) so that the cartridge isin the form of a disposable capsule cartridge.

FIG. 6 shows another device embodiment. This design is also shown inU.S. Pat No. 4,895,806 and consist of two halves, 70 and 72, the firsthalf 70 having an inlet 74 and the second half 72 having an outlet 76.Inside the device are a plurality of media discs 78 stacked one on topof the other between a top and bottom porous substrate 80 and 82respectively. Sealing around the circumference of the discs and the topand bottom of the stack of discs is provided by a series of gaskets 84.

FIG. 7 shows a variation on the design of FIG. 6 in which a series ofmedia layers 90 are separated from each other by spacers 92 and are allliquid tightly sealed around the top and bottom circumferences of seriesof media layers 90 to the interior of the device by gaskets 94. Thedevice is formed of two halves 98 and 100 with an inlet 102 and outlet104 formed in respective ends of the two halves 98 and 100 of thedevice.

FIG. 8 shows another device format formed of a cartridge 110 having aninlet 112 in one end 113 and an outlet 114 in the other end 115 and aseries of layers of media 116 in between. Porous spacers, 118 and 120are adjacent the inlet and outlet respectively and maintain the media inplace. The media is formed of a size such that the media contacts theinner wall of the device so that all flow must be through the body ofmedia rather than by passing the media along the inner wall or the like.

An additional embodiment of the present invention is to selectively coatonly certain areas of a substrate with the coating of the presentinvention. This can be accomplished through the use of masks, tapes,screens, soluble polymers and the like that are set done in apreselected pattern so only the uncovered areas are coated. The use ofpatterns such as grids, circles, squares and the like are useful inthese embodiments.

In FIG. 9 is shown another embodiment of the present invention. Asshown, there is a porous substrate 150 that has one or more areas ofcoated materials 152 and one or more areas 154 that are not coated. Inthis arrangement, the non-coated area(s) 154 are formed as a seriesintersecting lines that form a series of square grids, the interior ofeach square are formed of coated area 152. Such gridded structures areuseful in many applications such as in the analysis of proteins, DNA influids or as individual electrophoresis gels.

An alternative design is shown in FIG. 10, where the one or more coatedareas 160 of the substrate 162 are circular in design and set out ineven rows and columns. The area between the coated areas is a non-coatedportion 164 of the substrate. Such a design is useful in protein captureand other small volume separations. It may also be incorporated as thefilter in a multiwell plate with the coated areas aligning with the openbottom of the wells. The substrate can be attached to the plate as asingle sheet of material and have the coating only where there is activefiltration or flow. Optionally, the uncoated areas can be renderednon-porous or removed after attachment of the substrate to the wells tolimit cross contamination (cross talk).

Other device designs can and are possible with the present inventionincluding but not limited lenticular devices such as are shown in WO01/83077A1.

This invention also allows for the polymeric adsorptive layer tofunction without the additional requirement of the mechanical stabilityof the adsorptive layer needed to handle flow rates and pressure dropsassociated with a packed bed of beads. The hydrogel mechanicalproperties are “decoupled” from the mechanical requirements of thepacked chromatographic bed, because the porous substrate provides therequired stability. In this way, the inverse relationship betweenpressure drop and separation resolution associated with bead media canbe overcome. The porous material can consist of any porous material withthe correct pore size and pore size distribution. The pore sizedistribution must be reasonably narrow to prevent large variations inliquid flow velocities through the material (this could adversely affecta desired chromatographic separation). Reduced plate heights of ˜<0.1 cmare within the range of reasonable “pore size distribution”. In thisway, the diffusional pathway for an adsorbing entity will be shortenough so that diffusion into and out of the hydrogel will not be thelimiting parameter during the chromatographic separation as it is incurrent preparative scale bead media.

The media of the present invention can be used in lieu of conventionalchromatography media to capture the selected molecule from a streamcontaining it along with other molecules and contaminants. It may beused in a primary clarification step in which relatively unfiltered cellbroths; lysed cell broths and other crude process streams are initiallytreated to remove the larger contaminants. It may be used to treat bloodor other bodily fluids to remove the selected molecule, be it acontaminant or undesired entity such as a pathogen or leukocyte or adesired molecule such as a growth hormone or the like. It may be used inclassic chromatography application to purify proteins and othermolecules. It may also be used to remove viruses, endotoxins and otherimpurities before a final release of the product. As can be appreciatedthe media of the present invention can be tailored to fit almostparticular application or use.

EXAMPLE 1 Room Temperature Stable Agarose Solution Suitable for Coating

Six grams of agarose powder (type XII, obtained from Sigma-Aldrich) wereadded to 40 grams of water, the mixture was agitated while heating at atemperature of 95° C. until an initial agarose solution was formed. Thisinitial free flowing solution was cooled to room temperature, at whichpoint the solution became a gel having no free flowing characteristicsat all. To this gel, 15 grams of zinc chloride were added and themixture was heated again to 95° C. while agitating until the gel and thesalt dissolved to form a homogeneous solution. This solution was thencooled to room temperature; the solution's free flowing characteristicswere retained at this temperature. To this solution, 39.9 grams ofmethanol and 0.1 grams of Fluorad FC-95 fluorosurfactant (3M Company)were added while mixing to form the final agarose solution. This finalsolution remained liquid at room temperature.

EXAMPLE 2 Coating Using Room Temperature Stable Agarose

A polyolefin non-woven fabric having a pore size of about 100 micronsand a porosity of about 65% (Type FO2463 from Freudenberg of Lowell,Mass.) was coated with the agarose solution of Example 1 according tothe following procedure. The fabric was exposed to the agarose solutionof Example 1 such that the fabric was completely wetted by the solution.The wet fabric was then placed between two sheets of polyethylene filmand squeezed gently to remove excess solution from the surface of thefabric, the fabric was then removed from the film sheets and allowed todry at room temperature to remove the methanol and unbound water byevaporation. The dry coated fabric was then immersed in acetone to gelthe agarose and to remove the salt and surfactant thus creating thecoating of essentially pure agarose. The coated fabric was immersed inwater to further rinse the fabric and to remove the acetone, the agarosecoated fabric was then kept in water.

EXAMPLE 3 Crosslinking of Agarose Coating

The water-wet agarose coated fabric from example 2 was immersed in amixture containing 5 grams of epichlorohydrin and 95 grams of 2M sodiumhydroxide, the temperature of this mixture was then raised to 50° C. andthe crosslinking reaction was allowed to proceed at this temperature for16 hours under gentle agitation. The crosslinked coated fabric wasrinsed with water several times to remove excess reactants and base.

EXAMPLE 4 Functionalization of Crosslinked Agarose Coating withSulfopropyl (SP) Groups

The crosslinked agarose coated fabric of example 3 was immersed in asolution containing 6 grams of sodium bromopropanesulfonate 94 grams of1M sodium hydroxide, the temperature of this solution was then raised to50° C. and the functionalization reaction was allowed to proceed at thistemperature for 16 hours under gentle agitation. The sulfopropylfunctionalized coated fabric was rinsed with water several times toremove excess reactants and base, the fabric was kept in water. Thepermeability of the modified fabric was measured to be 1.78 cm²/min-psiin a sodium in a sodium acetate buffer at pH 4.5 and conductivity of 8mS.

EXAMPLE 5 Protein Binding of SP Functionalized Agarose Coated Fabric

A 13 mm disk of the SP functionalized agarose coated fabric from example4 was immersed in 6 ml of phosphate buffer at pH 7, conductivity of 2 mSand containing lysozyme in a concentration of 1 g/L, the fabric wasallowed to remain in contact with the protein solution for 16 hours atroom temperature under agitation. After 16 hours, the concentration oflysozyme in the protein solution was measured and the amount of proteinbound to the fabric was calculated based on the volume of the 13 mm diskof fabric. The protein binding capacity of the fabric was measured to be50mg lysozyme/ml fabric. The water flow rate through the media wasdetermined by measuring the flow rate through a circular sample of themodified fabric having a diameter of 13 mm and using a column of water15 cm in height. The sample had a flow rate of water of 50 ml in 14seconds under these conditions. The uncoated substrate had a flow rateof 50 ml in 6 seconds under the same conditions.

EXAMPLE 6 Cellulose Coating on Substrate

A polyolefin non-woven fabric of Example 2 having a pore size of about100 microns and a porosity of about 65% was coated with agaroseaccording to the following procedure. The fabric was exposed to asolution containing 3 grams cellulose and 97 grams ofN,N-dimethylacetamide saturated with lithium chloride, such that thefabric was completely wetted by the solution. The wet fabric was thenplaced between two sheets of polyethylene film and squeezed gently toremove excess solution from the surface of the fabric, the fabric wasthen removed from the film sheets and immediately immersed in water for10 minutes to form the coating. The cellulose coated fabric was kept inwater. The coated fabric was crosslinked and SP functionalized followingthe procedures of Examples 3 and 4. The lysozyme binding capacity of themodified fabric was measured according to the procedure of Example 5.The lysozyme binding capacity of the coated fabric was measured to be120 mg/ml fabric.

EXAMPLE 7 Dextran Coating on Substrate

A polyolefin non-woven fabric of Example 2 having a pore size of about100 microns and a porosity of about 65% was coated with dextranaccording to the following procedure. A coating solution was preparedcontaining 15 g dextran (500,000 MW), 1 g polyethylene glycol diglycidylether, 20 g polyacrylamide solution (10% in water), 1.5 g 1N sodiumhydroxide and 62.5 g water. The non-woven fabric was exposed to theabove dextran coating solution such that the fabric was completelywefted by the solution. The wet fabric was then placed between twosheets of polyethylene film and squeezed gently to remove excesssolution from the surface of the fabric, the fabric was then removedfrom the film sheets and allowed to dry at room temperature. The dry,coated fabric was then placed in an oven at 85° C. for 4 hours to effectcrosslinking of the dextran. The coated fabric was then rinsed in waterseveral times to remove any unreacted materials, including thepolyacrylamide. The coated fabric was kept in water. The crosslinkeddextran coated fabric was then SP functinalized according to theprocedure of Example 4 and the lysozyme binding capacity was measuredaccording to the procedure of Example 5. The lysozyme binding capacityof the coated fabric was measured to be 28 mg/ml fabric.

EXAMPLE 8 Polyallylamine Coated Substrate

A reaction medium having a pH of 9 was prepared using the followingformulation:

11.6% polyallylamine

11.6% sodium chloride

23.2% polyethyleneimine epichlorohydrin-modified (17% solution in water)

18.6% sodium hydroxide (1.0 N solution)

34.9% water

A 0.45 μ ultrahigh molecular weight polyethylene (UPE) membrane wasprewet with methanol and directly contacted with the above solution forabout 5 minutes. The wet membrane was placed in a polyethylene film bagand the bag was placed in an oven set at 85° C. for 7 minutes whilebeing careful not to dry out the membrane to initiate the crosslinkingreaction. The wet membrane was then removed from the bag and allowed todry at room temperature. The dry membrane was then placed in an oven at100° C. for four hours to complete the crosslinking reaction. Themembrane was then thoroughly washed with water, methanol andhydrochloric acid (1.0 N) and allowed to dry at room temperature. Themembrane was found to wet with water completely and had a strongaffinity for Ponceau—S, an anionic dye.

EXAMPLE 9 Monomeric Coating

A monomer solution was formulated as follows: 12.5g acrylamidopropanesulfonic acid, 1.875g methylene bisacrylamide, 6.8mL 50% NaOH, 3.8gsodium dodecylsulfate, 25g of polyethylene glycol (MW 8,000) and 76gwater. This solution was then diluted by 125mL of acetone with 0.5%Irgacure 2959 photoinitiator to form a coating solution. This solutionwas applied to a non-woven (Freudenberg FO2463) such that the non-wovenwas completely filled with the coating solution. The non-woven was thenair dried for 1-15 minutes to remove the majority of the acetone. Thenon-woven was then run through a UV initiation chamber at a frequency ofabout 200 to 450nm and an exposure time of about 2 seconds andpolymerization of the monomer solution occurred. The sample was thenwashed in a series (3) of water baths for 24 hours. The lysozyme bindingcapacity of the coated fabric was measured to be 64mg/ml fabric.

EXAMPLE 10 Monomeric Coating

A monomer solution of Example 9 was formulated except that it wasdiluted with 208mL of acetone containing 0.375% Irgacure 2959photoinitiator to form the coating solution. This solution was appliedaccording to Example 9. The lysozyme binding capacity of the coatedfabric was measured to be 68mg/ml fabric.

EXAMPLE 11 Monomeric Coating

A monomer solution of Example 9 was formulated except that it wasdiluted with 375mL of acetone containing 0.25% Irgacure 2959photoinitiator to form the coating solution. This solution was appliedaccording to Example 9. The lysozyme binding capacity of the coatedfabric was measured to be 70mg/ml fabric.

EXAMPLE 12 Monomeric Coating

A monomer solution of Example 9 was formulated except that nododecylsulfate was used in the coating solution. This solution wasapplied according to Example 9. The lysozyme binding capacity of thecoated fabric was measured to be 110mg/ml fabric. It had an ion exchangecapacity of 285 ┌eq/mL.

EXAMPLE 13 Monomeric Coating on Nylon Substrate

A monomer solution was formulated as follows: 12.5g acrylamidopropanesulfonic acid, 1.875g methylene bisacrylamide, 6.8mL 50% NaOH, 25g ofpolyethylene glycol (MW 8,000) and 80g water. This solution was thendiluted by 125mL of acetone with 0.25% Irgacure 2959 photoinitator forma coating solution. This solution was wicked into a woven nylon screen(Millipore Corp., 60 micron mesh) such that the non-woven was completelyfilled with the coating solution. The non-woven was then air dried for1-15 minutes to remove the majority of the acetone. The non-woven isthen run through a UV initiation chamber at a frequency of about 200 to450nm and an exposure time of about 2 seconds and polymerization of themonomer solution occurred. The sample was then washed in a series (3-5)of water baths for 24 hours. The resulting woven had an adsorptivecoating thickness of ˜10-30 microns as determined by optical microscopy.The lysozyme binding capacity of the coated fabric was measured to be67mg/ml fabric.

COMPARATIVE EXAMPLE 1 Agarose Coating According to WO 00/44928 onSubstrate

An 8M urea solution in water was prepared by dissolving 24.4 g urea in50 ml water. A 2.5% (w/v) solution of agarose was prepared by dissolving1.25g agarose (type XII, obtained from Sigma-Aldrich) in 50 ml of the 8M urea solution.

A polyolefin non-woven fabric of Example 2 having a pore size of about100 microns and a porosity of about 65% was modified with agaroseaccording to the following procedure. The fabric was exposed to theabove agarose solution in 8M urea for five minutes such that the fabricwas completely wetted by the solution. The wet fabric was then removedfrom the agarose solution and immediately placed in deionized water for24 hours. The modified fabric was then kept in water. The water flowrate through the fabric was determined by measuring the flow ratethrough a circular sample of the modified fabric having a diameter of 13mm and using a column of water 15 cm in height. The sample did not showany measurable flow under these conditions, suggesting that the pores ofthe fabric were substantially plugged by the agarose.

The advantages and results achieved through practice of the presentinvention include a novel composite media for separations where thebinding capacity is greater than the surface binding capacity (capacitybased on surface area, monolayer binding) but the diffusional distancerequired for an adsorbing entity to travel is sufficiently short so itdoes not hinder the mass transport. Therefore, the dynamic bindingcapacity is essentially independent of the convective flow rate; a novelmedia where the adsorbing/interacting material does not determine themechanical properties of the chromatographic bed.

The “decoupling” of mechanical and chromatographic performance allowsfor a larger optimization window for media development; a novel mediawith high resolution bead performance (similar to a =30μm bead), butwith a permeability similar to or greater than a preparative scale beadmedia (90μm bead). A high resolution preparative media for theseparations of biomolecules and other entities of interest; a novelmedia where the binding capacity is greater than the binding capacityexpected based on the surface area of the porous substrate; a novelmedia that does not require packing of the media before use. (As istypically required with bead materials) The composite porousmaterials/adsorbent hydrogel material can be fabricated into a“cartridge” device as thus not require packing prior to use and a novelmedia that could be disposable. The media cartridge could be easilydisconnected and disposed of without an “unpacking” procedure.

1. A porous coated media for adsorption or chromatography basedseparations comprising a base substrate, the base substrate being aporous, self-supporting structure formed of a non-woven fabric, and onemore porous polymeric coatings being formed of dextran and having athickness of from about 1 to about 30 microns on at least a portion ofall surfaces of the substrate, the one or more porous coatings having avoid volume fraction of the substrate of at least 1% and being porous tobiomolecules by adsorption in its bulk.
 2. The substrate of claim 1wherein the one or more coatings are formed on substantially all thesurfaces of the substrate.
 3. The substrate of claim 1 wherein thesubstrate has a pore size of from about 30 to about 200 microns.
 4. Thesubstrate of claim 1 wherein the coating has a thickness of from about 2to about 30 microns.
 5. The substrate of claim 1 wherein the coating hasa thickness of from about 2 to about 30 microns and an average pore sizefrom about 1 to about 200 nanometers.
 6. The substrate of claim 1wherein the substrate has a pore size of from about 50 to about 300microns.
 7. The substrate of claim 6 wherein the substrate has a poresize of from about 50 to about 200 microns.
 8. The substrate of claim 1,wherein the coated substrate comprises a fractional porosity of fromabout 0.35 to about 0.55.
 9. The substrate of claim 1 wherein thedextran coating has a thickness of from about 2 to about 15 microns. 10.A porous coated substrate for adsorption based separations comprising abase substrate, the base substrate being a porous, self-supportingstructure formed of a non-woven fabric having a pore size of from about30 to about 200 microns, and one or more porous polymeric coatings onall surfaces of the substrate, the one or more coatings being formed ofdextran having a thickness of from about 1 to about 30 microns andoccupying from about 1 to about 50% preferably from about 10% to about50% of the void volume of the pores of the substrate and the substratehas good convective flow and diffusional flow.
 11. The substrate ofclaim 10 wherein the dextran coating has a thickness of from about 1 toabout 20 microns.
 12. A porous coated substrate for adsorption basedseparations comprising a base substrate, the base substrate being aporous, self supporting structure formed of non-woven fabrics of plasticand one or more porous polymeric coatings on all surfaces of thesubstrate, the one or more polymeric coatings being formed of dextranhaving a thickness of from about 2 to about 30 microns and volume of thepores of the substrate, and the substrate having good convective flowand diffusional flow, an average pore size from about 1 to about 200nanometers, and occupying from about 10 to about 50% of the void. 13.The substrate of claim 12 wherein the dextran coating has a thickness offrom about 2 to about 15 microns.
 14. A porous coated substrate foradsorption based separations comprising a base substrate, the basesubstrate being a porous, self supporting structure being made fromthermoplastic non-woven fabrics and one or more porous polymericcoatings having a thickness of from about 1 to about 30 microns onsubstantially all surfaces of the substrate, the one or more coatingsbeing formed of dextran and occupying from about 10 to about 50% of thevoid volume of the pores of the substrate, and the substrate having goodconvective flow and diffusional flow.
 15. The substrate of claim 14wherein the dextran coating has a thickness of from about 1 to about 20microns.
 16. The substrate of claim 14 wherein the dextran coating has athickness of from about 2 to about 15 microns.
 17. A porous coatedsubstrate for adsorption based separations comprising a base substrate,the base substrate being a porous, self supporting structure formed of anon-woven fabric structure formed of a non-woven fabric having a poresize of from about 30 to about 200 microns, and one or more porouspolymeric coatings being formed of dextrin having a thickness of fromabout 1 to about 30 microns on all surfaces of the substrate and havingan average pore size of from about 1 to about 100 nanometers, the one ormore coatings occupying from about 1 to about 50% of the void volume ofthe pores of the substrate and the capacity of the substrate isindependent of the convective flow properties of the substrate.
 18. Thesubstrate of claim 17 wherein the dextran coating has a thickness offrom about 2 to about 15 microns.
 19. A porous coated substrate foradsorption based separations comprising a base substrate, the basesubstrate being a porous self supporting structure being made fromnon-woven fabrics having a pore size of from about 30 to about 300microns, and one or more porous coatings being formed of dextran andhaving a thickness of from about 2 to about 30 microns on substantiallyall surfaces of the substrate, wherein the coated substrate comprises afractional porosity of from about 0.35 to about 0.55.
 20. The substrateof claim 19 wherein the dextran coating has a thickness of from about 2to about 15 microns.